Cell, Vol. 61, 397-405, May 4. 1990, Copyright 0 1990 by Cell Press

Dominantly Inherited Expre on of BID, an Invariant Undiversified T Celi Receptor 6 Chain

Gek-Kee Sim’ and Andrei Augustint l Base1 Institute for Immunology Grenzacherstrasse 487 CH-4005 Base1 Switzerland t Department of Medicine National Jewish Center for Immunology

and Respiratory Medicine Denver, Colorado 80206 and Department of Microbiology and Immunology University of Colorado Health Sciences Center Denver, Colorado 80262

Summary

In BALBlc lung and lymph node y6 T cells, a large frac- tion of the expressed Vh5 genes consist of an invari- ant sequence, BID (for BALBlc invariant delta). BID results from a direct joining of the Vb5, D6*, and JgI segments, which conserve their complete germline coding sequences. In C57BL/6 (H-2b) mice, where identical and functional segments are present in the germline, BID is absent. It appears that BID+ y6 T cells am positively selected by factors encoded outside of the classical MHC region, as indiiated by their domi- nance in Fl(C57BU6 x BALBIc) and in BALRB (H-2b) mice. Additional observations, including the expres- sion of BID in BALB/c nulnu but not in C57BU6 nulnu mice, suggest that the expansion of BID+ T cells es- sentially occurs extrathymically.

Introduction

T lymphocytes may express one of two types of antigen receptors: up or yS Both T cell receptors (TCRs) are so- matically assembled via rearrangements from immuno- globin-like genetic structures (reviewed in Tonegawa, 1983; Davis and Bjorkman, 1988). A large number of germline gene elements contribute toward the high de- gree of variability observed for a6 TCRs. This type of receptor is expressed on the majority of T cells in lym- phoid organs. a8 T cells are well-characterized helper and cytotoxic lymphocytes, known to recognize antigens only in the context of major histocompatibility complex (MHC) gene products. In contrast, the in vivo function of yS T cells is not known (reviewed in Raulet, 1989). Recent work on yS T cell clones has indicated that at least some yS T cells can recognize MHC gene products (Matis et al., 1987; Rivas et al., 1989; Haregewoin et al., 1989) while others appear to see class l-like antigens encoded in the Qa or TL regions (Bluestone et al., 1988; Bonneville et al., 1989; Vidovic et al., 1989). There are also reports of yS T cells that recognize mycobacterial antigens in the absence of MHC presentation (Holoshitz et al., 1989; Janis et al., 1989; O’Brien et al., 1989).

In the mouse, yS T cells constitute a minor population

in lymphoid organs, but appear in significant proportions in some nonlymphoid tissues such as skin, intestine, and lung (Bluestone et al., 1987; Koning et al., 1987; Goodman and LeFrancois, 1988; Augustin et al., 1989). A potentially very large repertoire of yS TCRs can be generated from a rather limited number of germline gene segments by junctional diversification (Elliott et al., 1988). A single VS gene may associate with two D elements simultaneously, and a number of random nucleotides may be added at one to three junctional points. Experimental data accumulated to date indeed show that a large number of..distinct and very diverse sequences can be displayed by yS TCRs (El- liott et al., 1988; Takagaki et al., 1989a, 1989b). Nonethe- less, the repertoire of yS clonotypes appears to be signifi- cantly affected by two factors. First, the rearrangement of individual y and S V genes apparently follows a defined program in ontogeny in the developing thymus (Garman et al., 1986; Havran and Allison, 1988; ho et al., 1989). Second, in several epithelial organs, a specific subset of V, and/or V6 genes is preferentially expressed (Asarnow et al., 1988, 1989; Takagaki et al., 1989b). In the case of dendritic epidermal cells (dECs) in the skin, identical rear- rangements with similar junctional sequences seem to give rise to a monotonous repertoire. The mechanisms governing this peculiar selection and distribution of yS clonotypes at various anatomical sites are still poorly un- derstood.

Recently, we have identified a set of yS T cells residing in the murine lungs (Augustin et al., 1989). A large fraction of these cells respond to mycobacterial antigens in vivo as well as self heat shocked cells in vitro (Augustin et al., 1989; Rajasekar et al., 1990). In the hope of learning more about the biology of yS T cells, we have started to charac- terize the repertoire of the yS TCRs expressed among mu- rine resident pulmonary lymphocytes (RPLs) with respect to their mRNA sequences. In doing so, we have discov- ered a peculiar Vss rearrangement in BALB/c mice that is distinguished by an invariant VDJ junction. This specific VDJ rearrangement accounts for the major species of Vh5, expressed in BALB/c mice but absent in the C57BU6 strain. By taking advantage of this observation, we have determined that T cells expressing the sequence BID (for BALBlc invariant delta) are selected in the murine immune system according to rules partially distinct from those cur- rently accepted for the selection of the a6 repertoire.

Results and Discussion

The VP Repertoire of BALB/c RPLs Is Highly Diverse The repertoire of epithelium-associated yS T cells may be highly diversified as in the intraepithelial lymphocytes (IELs) of the intestine (Takagaki et al., 1989; Asarnow et al., 1989) or very limited as in the dECs of the skin (Asar- now et al., 1988). To estimate the degree of yS TCR diver- sity in RPLs, we first determined the extent of various V6 gene usage in an RPL population isolated from normal, unimmunized BALBlc mice. yS T cells were prepared from

Cell 398

Figure 1. TCR V6 Gene Expression in RPLs

VS cDNA was synthesized from RNA of BALD/c RPLs, and each V gene sequence was specifically amplified by PCR as in Experimental Procedures. After 12 cycles of amplification, 5 ul of each reaction prod- uct was loaded onto a 1.5% agarose gel, electrophoresed. transferred onto a nylon membrane, and hybridized to a JB1 probe. The autoradio- gram shown represents a 1 hr exposure with intensifying screens. Mo- lecular weight standards from Alul-cleaved PER322 DNA are marked. Only molecules of the appropriate sizes hybridized.

the lung as previously described (Augustin et al., 1989) and the relative abundancies of the various V6 RNAs in the population were estimated by Southern blot analysis of polymerase chain reaction (PCR)-amplified S cDNAs. As shown in Figure 1, the major V6 gene transcripts pres-

V6 Nl DS 1 N2

GERMLINE GTGGCATATCA

vs3 : 203-2 TGT ATC CTG AGA G CCCG ATATC C

ent in RPLs appear to be derived from the Vs6 gene fam- ily. VsS is also expressed at a significant level, while Vs7 and Vg4 mFiNAs are infrequent but clearly discerned. V6, and Vs3 transcripts are barely present, and no apparent Vs2 transcripts are detectable. This pattern of VS gene ex- pression is reminiscent of that in IELs, the main difference being that Va6 and Vs7 predominate in IELs, while Vs6 and VsS constitute the major species in RPLs. It should be noted that the hybridization probe in Figure 1 is a JsI probe. On a duplicate filter probed with Js2, no detectable signal is observed (data not shown). This, together with the fact that we have not found a single Js2 containing cDNA clones among those that we have sequenced, ar- gues against the use of Js2 in any significant fashion in BALB/c RPLs.

In the case of yS TCR genes, the detection of a correctly sized transcript is by no means synonymous with receptor expression. Nonfunctional mRNAs, resulting from rear- rangements that generate out-of-frame or prematurely terminated translation products, are quite frequent. We therefore performed DNA sequence analysis on the cloned PCR products in order to confirm the pattern of V gene us- age observed and to determine the extent of junctional diversity associated with these V genes. With the excep- tion of Vsr, the vast majority of cDNA clones can code for functional polypeptides. All the Vsl cDNAs we have se- quenced, however, carry frame-shift mutations. The data presented in Figure 2 show only in-frame sequences that can be translated into receptor chains. In almost all cases examined, we observe the presence of the “adult” type 6

D6 2 N3 J8 1

ATCGGAGGGATACGAG CT ACC GAC AAA

TCGGAGGG TTG CT ACC GAC AAA 203-5 TGT ATC CTG AG AG TA GCT TCGG AATT T ACC GAC AAA 203-6 TGT ATC CTG AGA G T GCAT GA CGGAGGGATACGAG CCCCG ACC GAG AAA 203-12 TGT ATC C GT T ATCGGAGGG CCG CT ACC GAC AAA

v6 4 : 204-2 TGT GCT CTC ATG GA TCGGAGGGA CG CT ACC GAC AM 204-3 TGT GCT CTC ATG GAG C TGA TGG GGTGCCCAT ATCGGAGGGATA TCCCTC CC GAC AAA 204-5 TGT GCT CTC ATG GAG CG TGG ATCGGAGGGATACGAG C CT ACC GAC AAA 204-E TGT GCT CTC ATG GAG CG GG GTGGCAT GC CC GAC AAA 204-9 TGT GCT CTC ATG G TG cc ATC CCGTTGC ACC GAC AAA

VS6 : 206-l TGT GCT CTC TGG GAG CT GGA CGGAGGGATACGAG TTTCCC CT ACC GAC AAA 206-2 TGT GCT CTC TGG GAG CT GTGGCATATCA G GGAGGGATACGAG CTCCGG ACC GAC MA 206-3 TGT GCT CTC TGG GAG CT AGCGGG CGGAGGGATACGAG CT ACC GAC AAA 206-10 TGT GCT CTC TGG GAG C CCAC GTGG AC CGGAGGG CCCG CT ACC GAG AAA 206-5 TGC GCT CTC TCG GAA CT G ATCGGAGGGATACGAG CT ACC GAC AAA 206-9 TGC GCT CTC TCG G CCTTCTTT ATCGGAGGGATACG TGCTTGGG CT ACC GAC AAA 206-12 TGC GCT CTC TCG GZA CT GA AT G GGGAT CCCG CT ACC GAC AAA

vs7 : 207-2 TGT GCT ATG G GCCCGCC GAGG ACC GAC AAA 207-5 TGT GCT AT AGAT ATCGGAGGGATACGAG CT ACC GAC AAA 207-9 TGT GCT ATG G A TGG G GGGATACGAG CT ACC GAC AAA 207-10 TGT GCT ATG G ATCCCAT ATCGGAGGGATACGAG CTAGACT CT ACC GAC AAA 207-11 TGT GCT ATG G AA TGGCA C CGAG CT ACC GAC AAA

Figure 2. A High Degree of Junctional Diversity Is Associated with Most V6 Genes Expressed in RPLs

A sample of DNA sequences at the VDJ junctions of V6 gene transcripts isolated from BALB/c RPLs is shown. Only sequences that represent trans- latable mRNA are presented.

Positive Selection of $5 T Cells 399

BALB/C RPL :

A) v6 5 Nl D61

GERMLINE TGT GCC TCG GGG TAT. GTGGCATATCA

BID TGT GCC TCG GGG TAT

BID-LIKE: 105.65 TGT GCC TCG GGG TAT 105.31 TGT GCC TCG GGG TAT 105.211 TGT GCC TCG GGG TAT 105.33 TGT GCC TCG GGG TAT

Others: 105.7 TGT GCC TCG GGG AA GGCAT 105.62 TGT GCC TCG GGG GCATAT 105.23 TGT GCC TCG GGG TAT ATTC 105.28 TGT GCC TCG GGG cc GTGGCATATC

(B) V65 Nl/Dl/NZ/DZ/NS

germline CASGY IGGIRA

BID CASGY IGGIRA

BID-like 105.65 CASGY IGGIRS 105.31 CASGY IGGIRV 105.211 CASGY IGGIR 105.33 CASGY IGGIRA

Others: 105.62 CASG AYTGGIRVA 105.7 CASG KAYRRDTS 105.23 CASGY ILGGIRAP 105.28 CASG PWHILSGGIRA

N2 06 2

ATCGGAGGGATACGAG

ATCGGAGGGATACGAG

ATCGGAGGGATACG ATCGGAGGGATACGAG ATCGGAGGGATACG

ATCGGAGGGATACGAG AC CGGAGGGATACGAG

TCGGAGGGATACGAG CTCTC CGGAGGGATACGAG

J61 TDKL

TDKL x15

TDKL DKL

TDKL x2 DKL

TDKL DKL DKL DKL

N3 56 1

CT ACC GAC AAA

CT ACC GAC AAA x15

TT CT ACC GAC AAA T C GAC AAA G ACC GAC AM X2

CC GAC AAA

C GAC AAA TAG CT ACC GAC AAA CTC CC GAC AAA

CT ACC GAC AAA

Figure 3. Preferential Expansion of the Undiversified Sequence BID among V&s Chains in BALB/c RPLs

(A) The VDJ junctional regions of BID and BID-like sequences are shown, together with the highly diversified junctional regions of a contrasting group of independent V&s rearrangements, all of which are productive. The data are combined from three independent experiments, each of which reflects a predominant BID population, the presence of BID-like sequences, and a minor fraction of highly diversified V&s rearranm transcript. (B) The predicted amino acid sequences of the VDJ junctions of the V&s clones shown in (A). The two R’s in bold type have been regenerated by DJ junctional diversification

(Elliott et al., 1988; Lafaille et al., 1989) involving exten- sive N region additions of random nucleotides and/or the simultaneous use of two D elements in conjunction with JsI. In addition, no two identical sequences have been found. Overall, these data demonstrate that the yS TCR repertoire of RPLs, like that of IELs, is potentially rather large. An unusual exception, Vs5, is presented separately in Figure 3A and discussed in the next section.

The Major Vb5 Clonotype in BALBlc RPLe Is Encoded by BID, an Invariant Vs5D~& TCR Sequence In the first set of analyzed Vse-positive cDNA sequences of BALB/c RPL origin, a recurrent pattern of rearrange- ment was detected. Two additional experiments starting from independent sets of BALB/c mice were performed to investigate whether a conserved sequence was indeed dominant among Vs5 mRNA species in y6 T cells of lung

origin. Data obtained from these three experiments are presented in Figure 3. The invariant sequence BID has been found in all three experiments, at frequencies of 8/9, 4/9, and 3/6 among in-frame sequences. It has several striking structural characteristics: perfect nucleotide se- quence identity among the independently isolated clones from the different experiments; a unique V~sDr&, rear- rangement in which all the assembled segments retain

their entire germline structures; no apparent N nucleotide additions at the VD or DJ junctions; and the presence of the short palindromes ATAT and AGCT at the joints of VD and DJ, respectively. These short palindromes are reminiscent of the P dinucleotides (Lafaille et al., 1989) but are probably germline sequences.

Although not identical, five additional sequences (Fig- ure 3A) are similar to the BID prototype: one of them (105.211) has been found independently in experiments 1 and 3, while the others (105.65, 105.31, and 105.33) are unique. Like BID, they all display the unaltered ATAT palin- drome at the VD joints. However, a few insertions occurred at the site of the nucleotide deletions at the DJ junction. At most two nucleotides have been deleted from the 3’end of the germline Db2 elements, while as many as four nucleotides have been deleted from the 5’ end of JsI. The net consequence is that their predicted amino acid se- quences are either of the same length or one amino acid shorter than BID (Figure 38). In all cases, five of the six Ds2 germline-encoded amino acids have been retained, and in two cases, the fifth amino acid was generated by independent conservative nucleotide substitutions at the third position of the codon. The existence of this group of BID-like sequences strongly supports the idea that BID has been selected for in the generation of the BALBlc yS TCR repertoire and that the amino acid sequence at the

Cell 400

vs 5 Nl 061 N2

GERMLINE TGT GCC TCG GGG TAT. GTGGCATATCA

C57B6/J RPL :

305-2 TGT GCC TCG GG AC GGCATAT GA 305-7 TGT GCC TCG AGC GGC GA 305-10 TGT GCC TCG GGG A 305-12 TGT GCC TCG GGG A ATAT GGCGCCCAT 305-19 TGT GCC TCG GGG T CCAAT 305-20 TGT GCC TCG GGG A GC 305-23 TGT GCC TCG GGG T GGGGGGT 305-25 TGT GCC TCG GGG CCCG 305-27 TGT GCC TCG GGG TCGGG 305-30 TGT GCC TCG GGG A GGC GT 305-32 TGT GCC TCG GGG GCCAAT 305-33 TGT GCC TCG GGG GT 305-35 TGT GCC TCG GGG TC :TGGGGGG GGCA AAT 305-45 TGT GCC TCG GGG T TGGCA 305-46 TGT GCC TCG G AC GTGGCATATC G 305-48 TGT GCC TCG GG TAAC GTGGC CTCTAT

C57B6/J THYMOCYTES :

KN6-~23 TGT GCC TCG GGG TAT TG KN6-p22 TGT GCC TCG G CGTCCC ATATC KN102D2 TGT GCC TCG KN106D2 TGT GCC TCG GGG ccc GTGGCATA CCCT KNlOSD3 TGT GCC TCG GGG GGACAG GTGGC TCCTT

C5786/J IEL :

pd517 TGT GCC TCG GGG TAT TGG ATATC pd586 TGT GCC TCG GGG TAT ccc GCAT pd51tl7 TGT GCC TCG GGG GAA ATAT pd5238 TGT GCC TCG GG CTTC (30) TGT GCC TCG GGG GAAA TGGCATAT ATAGG

Figure 4. Absence of BID in C57BU6 RPLs, IELs. and DN Thymocytes

D6 2

ATCGGAGGGATACGAG

GAGGGATACGAG ATCGGAGGG ATCGGA ATCGGAGGGATACG ATCGGAGGGATACGAG ATCGGAGGGATAC

GGG CGGAGGGATAC

ATCGGAGGGATACGAG ATCGGAGGGATACGAG ATCGGAGGG ATCGGAGGGATACGAG

CGGAGGGATACGAG ATCGGAGGGA ATCGGAGGGATACG

GGAGGGATACGAG GGAGGGAT

ATCGGAGGGATACGAG ATCGGAGGG ATCGGAGGGAT

GAGGGATACGAG CGGAGGGA

GG GAGGG

GGAGGGATACG

N3

CCCCCTCG AGGAAG GGCCCC CTC ccc

TCC

CCTCG GG AA CCTT CAC

CTG

CTG CCTT

CTTGT G CCCT CCCCA TAG

561

CT ACC GAC AAA

CT ACC GAC AAA CC GAC AAA

CT ACC GAC AAA CC GAC AAA CC GAC AAA

CT ACC GAC AAA T ACC GAC AAA

CT ACC GAC AAA CT ACC GAC AAA CT ACC GAC ALA CT ACC GAC AAA CT ACC GAC AAA

ACC GAC AAA CT ACC GAC AAA CT ACC GAC AAA

AC AAA

ACC GAC AAA ACC GAC AAA

CC GAC MA CT ACC GAC AAA

ACC GAC AAA

CC GAC AAA CT ACC GAC MA CT ACC GAC AAA CT ACC GAC AAA CT ACC GAC AAA

The IEL sequences are from Takagaki et al. (1989b) and Asarnow et al. (1969) and the thymocyte sequences are from Takagaki et al. (1989a). Only productive rearranged sequences are shown. The thymocyte sequences from Elliott et al. (1988) were not included because they were not obtained from C57BU6. However, there were no BID or BID-like sequences reported.

VD junction, extending through the fifth residue(R) of the D region (Figure 3B), is critical to the selection process. By inference, the exact nature of the amino acid residue at the DJ junction appears to be less crucial. One could speculate that the frequent presence of the alanine resi- due (A) at the DJ junction was more a consequence of the type of rearrangement mechanisms that are at work dur- ing the process of generating this particular BID rear- rangement instead of the nature of the amino acid itself.

One could also note that the remaining four Vs5 cDNA sequences are all unique and contain highly diversified VDJ junctions similar to those previously shown to be as- sociated with Vh5 by other investigators (Elliott et al., 1988; Takagaki et al., 1989a, 1989b). This type of junc- tional region diversity is also present in the other S vari- able region sequences used in RPLs (Figure 2). The pres- ence of this group of Vs5 cDNAs in BALBlc RPLs argues against the notion that the lung is populated with V65 receptors only at a time when the machinery for extensive N region diversification is not yet functional, as was postu- lated for the Vgl of dECs (Asarnow et al., 1988). In the case of dECs, it has recently been shown that 5 out of 12 in-frame Vy5 sequences found in adult thymocytes are of the dEC “fetal” type, suggesting that dECs continue to be generated and dispatched to the periphery well after birth and the onset of the mechanism for extensive N region di-

versification, in spite of the lack of N region diversity in this particular receptor. That BID predominates in the pres- ence of a rather large repertoire of other Vss rearrange- ments may be another indication of the selective pressure that maintains its status.

BID Is Not Detected in C57BU6 Mice We could not find the BID sequence among the published Vs5 clones (Elliott et al., 1988; Asarnow et al., 1989; Ta- kagaki et al., 1989a, 1989b). However, since most of them were of C57BU6 origin and none of them were isolates from BALB/c mice, we could not determine whether the high representation of this clonotype in pulmonary T cells was BALB/c specific, tissue specific, or both.

The published sequences, shown in Figure 4, reveal no BID or BID-like sequences in C57BU6 thymocytes of IELs. Since BID is present at a very high frequency among the Vss mRNAs isolated from BALB/c RPLs, we reasoned that perhaps BID was not detected by previous investiga- tions because it is specific to the lung tissue. We therefore amplified, cloned, and determined the junctional region sequences of the Vss mRNAs isolated from C57BU6 RPLs. Sixteen potentially functional sequences are pre- sented in Figure 4. They all exhibit a high degree of diver- sity at the VDJ joining regions, similar to those found in thymocytes and IELs from the same mouse strain. The

Positive Selection of y6 T Cells 401

vs 5 Nl 06 1 N2

GERMLINE TGT GCC TCG GGG TAT. GTGGCATATCA

(A) Fl (C57BL6/J X BALB/C) RPL:

BID TGT GCC TCG GGG TAT

BID-Like: SOS-3 TGT GCC TCG GGG TAT

Others: 505-Z TGT GCC TCG GGG TAT AC 505-5 TGT GCC TCG GGG TAT CCA GTGGCAT 505-a TGT GCC TCG GG CCCA 505-10 TGT GCC TCG GGG C 505-13 TGT GCC TCG GGG TAT GGC

(B) BALB.B WL:

BID TGT GCC TCG GGG TAT

BID-Like: 605-6 TGT GCC TCG GGG TAT

Others: 605-l TGT GCC TCG GG CCAT 605-7 TGT GCC TCG GGG CAT

Ds 2

ATCGGAGGGATACGAG

ATCGGAGGGATACGAG

ATCGGAGGGATACG

CGGAGGGATACG ATCGGAGGGA

GGAGGGATACGAG TCGGAGGGATACG

GGAGGGATACGA

ATCGGAGGGATACGAG

ATCGGAGGGATACGAG

ATCGGAGGGATACG ATCGGAGGGATACGAG

N3 561

CT ACC GAC AAA

CT ACC GAC AAA x6

GC CC GAC AAA

GG CT ACC GAC AAA T ACC GAC AAA

CT ACC GAC AAA CG cc GAC AAA CGC ACC GAC AAA

CT ACC GAC AAA x8

CC GAC AAA

CC GAC AAA CT ACC GAC AAA

Figure 5. BID Is Positively Selected in Fl(C57BU6 x BALE/c) and Is Predominant in BALE.6 (H-2b) Mice

(A) Junctional regions of expressed Vas genes in Fl(C57BU6 x BALBk) mice. (B) Junctional regions of expressed Vss genes in BALB.B mice. All sequences shown represent in-frame rearrangements.

BID prototype as well as the BID-like sequences, however, are conspicuously absent.

In light of the fact that the germline structural se- quences for BID are all present in C57BU6 as well as in BALB/c mice, these data strongly suggest that the expres- sion of BID is either forbidden in the C57BU6 strain of mice (negatively selected) or is positively selected for by an element in BALBlc absent in C57BU6. To date, it is not known whether y6 T cells are also subjected to these rules, which have been shown to apply to their a6 counter- parts. To obtain more information on this issue, the genetics of inheritance as well as the tissue distribution of the BID phenotype was further investigated.

BID Is Dominant in Fl (C57BU6 x BALB/c) and Is Also Expressed in BALB.B Mice To establish whether the high BID expression in RPLs is dominantly inherited, cDNA clones derived from a pool of Fl(C57BU6 x BALB/c) mice were subjected to sequence analysis. From Figure 5A, it appears that six BID+ cDNA clones and one BID-like sequence were found among the twelve in-frame sequences analyzed in this experiment. This result indicates that the high expression of BID mRNA in BALBlc mice is a dominant trait. Therefore, one is tempted to assume that the mechanism leading to the presence of frequent BID+ T cells could be described as “positive selection!’ In that case, data accumulated on the selection of af3 TCRs might suggest the involvement of the thymus in such selective events with the active participa- tion of some MHC allelic gene products, expressed on thy- mic epithelial cells. However, as apparent from Figure 5B, the BALB/c H-2 congenic BALB.B strain is positive for BID. BALB.B is thought to have derived its MHC haplotype from

the B.10 mouse, and therefore carries the H-2b MHC al- leles on a BALB genomic background. In 1BALB.B RPLs, eight BID sequences and one BID-like junctional region are present among the 11 in-frame Vh5 rearrangements analyzed. Thus, it appears that a structure or structures not encoded for in the classically defined BALB/c H-2d haplotype could be responsible for the dominant in- heritance of the BID phenotype in the Fl(C57BL/6 x BALB/c) mice.

Recent reports have indicated that an unusually high frequency of y6 T cells react to MHC class l-like antigens encoded by Qa and TLa loci. Since the exact crossover points that resulted in the generation of the BALB.B mouse are not known and the T region alleles of BALB.B have not been actually classified, we could not rule out the possibility that differences between BALB.B and C57BL16 in the TLa region are responsible for the observed differ- ence in BID expression. However, the Qa genes are prob- ably not involved in the selection of BID, since C57BL16 and BALBlc express the same Qa-1 allele (Klein et al., 1983), while BALB/cJ, which shares the same Qa-2 allele as C57BL16, is also BID positive.

The statistical significance of the data presented above was verified via Fisher’s exact test for independence. We scored as positive events in all cases only the strict BID sequences, even though there exist very similar “BID-like” sequences, which are present only in BALD/c, Fl(C57BU6 x BALB/c), and BALB.B RPL populations. Comparing the same populations on the basis of BID plws BID-like se- quences versus other sequences would yield yet lower p values (data not shown). Two by two comparisons, per- formed between all RPL sequences anafyzed, are pre- sented in Table 1, where it can be seen that at least two

Cell 402

Table 1. Probabilities that Pairs of RPL Populations Have the Same Frequency of BID Expression

Fi (C57BU6 C57BU6 x BALBk) BALB.B

BALBlc 0.3 x lo-da 0.4 0.4 C57BU6 - 0.2 x lo-2a 0.7 x 10-d = Fl(C57BU6 - - 0.2

x BALB/c)

Probabilities are given by Fisher’s exact test for independence in a two by two contingency table performed according to Steinberg (1965). a These values indicate significant differences.

orders of magnitude in the p value separate the BALB/c, Fl(C57BU6 x BALB/c), and BALB.B RPL populations on one hand, and the C57BU6 RPL population on the other hand, in terms of BID expression. That is, BID is expressed in RPLs of BALB/c, Fl(C57BU6 x BALBlc), and BALB.B, but not in C57BU6 RPLs.

Extrathymic Expansion of BID+ T Cells It has been shown in various experimental systems that an essential step toward the positive selection of various ap clonotypes takes place in the thymus (Teh et al., 1988; Kisielow et al., 1988; Sha et al., 1988; Berg et al., 1989; Kaye et al., 1989). The selected clonotypes are thus pres- ent both in the thymus and the periphery. In our ex- perimental system, an equivalent situation would be rep- resented by the presence of BID in the thymus of BALB/c mice. However, as shown in Figure 6, among 15 distinct in-frame Vgg cDNA sequences obtained from 4-week-old adult thymocytes, not a single one exhibits BID character- istics. One could argue that BID, owing to its lack of N re- gion diversity, is likely to represent a 6 sequence that had rearranged in early fetal life and thus could no longer be found in adult thymocytes. However, this assumption, which should be also applicable to the selection of dECs, appears less probable in light of the recent finding that 5/12 in-frame VYs sequences present in adult thymocytes are actually of the “fetal” dEC type (Lafaille et al., 1989). Moreover, V65 is known to be the predominant rearrang- ing g gene in the adult thymus (Elliott et al., 1988; Taka- gaki et al., 1989a), and its expression is at a relatively lower level among fetal thymocytes. If we assume that BID is expressed in the fetal thymus only, this would imply that BID is well maintained and expanded in the periphery of the permissive mice such that it still retains a dominant status in spite of the various other Vas rearrangements that are continuously being produced in the adult thymus.

To investigate the absolute requirement of the thymus for the positive selection of BID in BALB/c mice, we com- pared the frequency of BID among RPLs isolated from BALBlc nulnu and C57BU6 nulnu mice. This experiment was possible since the y5 T cells of pulmonary origin could be isolated from athymic nude mice and the pres- ence of the yS TCRs verified by immunoprecipitation (un- published data). As shown in Figure 6, BID is present in 719 of the productive cDNA sequences obtained from BALB/c nulnu RPLs, while it is absent among the 11

C57BU6 nulnu clones analyzed. The statistical analysis, performed as previously described, yields a value of p = 0.45 x 10e3, indicating that in athymic mice as well these differences are significant. That is, BALBlc nulnu RPLs are BID+ while C57BL16 nulnu RPLs are BID-. Thus, the predominance of BID is due to selective events that do not take place in the thymus. Taken together with our failure to detect BID in the BALB/c adult thymus, these data sug- gest that BID is expanded in the periphery and is probably even selected in a thymus-independent fashion.

To determine whether the peripheral expansion of BID can indeed continue long after the thymus has been depleted of potential BID sequences and to test whether BID can be present outside of the RPL set, we obtained the cDNA sequences of clones derived from systemic lymph nodes of 30-week-old BALBlc mice. Although the frequency of yS T cells is much lower in lymph nodes than in lungs, sufficient material was obtained from a large number of mice. These sequences, presented in Figure 6, display a clear predominance of BID among them. This in- dicates that BID is not present exclusively in the lungs and that it survives and remains dominant in the periphery for a long time, while the thymus produces BID- sequences. Obviously, this pattern of selection, in which the Fl is dom- inant but in which the peripheral expansion seems to play a preponderant role, while the thymus might play at best a minor part, is distinct from the one proposed to date for the selection of the a8 repertoire.

Concluding Remarks The mechanisms that govern the selection of the yS T cell clonotypes are poorly understood. However, they appear to be rather diverse. Some recurrent sequences of y or S chains described to date are not only of a so-called fe- tal type (i.e., they contain gene segments generally ex- pressed in the fetal thymus and their rearrangement intro- duced little diversity), but their localization, outside of the thymus, appears restricted to specific epithelial tissue. However, their structure is not strain specific in the mouse (Asarnow et al., 1988, 1989). Other y V genes, which ob- serve a specific organ distribution, display a large junc- tional diversity, the typical example being IELs, and no particular dominant phenotype has been found among them. Finally, several other V gene rearrangements, ex- pressed at specific times in development, do not have yet either a functional or topological assignment.

The invariant S chain that we isolated from the BALBlc mouse has several distinct properties. Although it dis- plays germline segments in their unaltered configurations and therefore represents a fetal type of rearrangement, it used JsI, which preferentially rearranges in the adult thy- mus (Lafaille et al., 1989). Vh5, the V gene used in BID, is abundantly expressed in the thymus and also at various extents in other organs such as lymph nodes, lungs, and intestines, in a variety of combinations with Da,, 0~. or both, giving rise to a high degree of diversity to which a large number of removed and added nucleotides contrib- ute. This image of diversity, present also in the C57BU6 RPLs, is drastically changed in BALB/c however, where BID is dominant in the periphery but not in the thymus.

Positive Selection of yS T Cells 403

vs 5

GERMLINE TGT GCC TCG GGG TAT.

BALB/c nu/au RPL: BID TGT GCC TCG GGG TAT 0ttEl-S: “5-l TGT GCC TCG GGG TAT “S-4 TGT GCC TCC GGG C57BL6/J nu/.u P.PL: 705-4 TGT GCC TCG GGG TAT 705-6 TGT GCC TCG GGG TAT 705-6 TGT GCC TCG GGG TAT 705-11 TGT GCC TCG GGG T 705-12 TGT GCC TCG GGG TAT 705-14 TGT KC TCG GGG TAT 705-15 TGT GCC TCG GGG TAT 705-16 TGT GCC TCG 666 T

705-20 TGT GCC TCG GGG 705-22 TGT KC TCG GGG 705-23 TGT GCC TCG GGG TAT

B&LB/C LYMPB liom : BID TGT GCC TCG GGG TAT BID-Like: 805.9 TGT GCC TCG GGG TAT Other*: 805.11 TGT GCC TCG GGG BALB/c TBYb,OCYTEI : 405-T TGT GCC TCG GGG TAT 405-8 TGT GCC TC 405-13 TGT GCC TCG GGG 405-16 TGT GCC TCG 666 T 405-20 TGT GCC TCG GGG 405-23 TGT GCC TCG GCG T 405-25 TGT GCC TCG GGG 405-29 TGT GCC TCG GGG 405-31 TGT GCC TCG GGG 405-36 TGT GCC TCG GG 405-37 TGT GCC TCG GGG T 405-39 TGT GCC TCG GG 405-42 TGT GCC TCG GGG TAT 405-43 TGT GCC TCG GGG TAT 405-45 TGT GCC TCG GG

Nl

c TCACCC

CTCT ATT ATTC CTAC A G

GGCAT

ccc AG

TGCA

AT TAC *ccc cc G CTC AC GGGC AA AC TC TT

OS 1 GTGGCATATCA

GGCAT GTGGC

GTGGCATRT GTGGCA

TGGC GTGGCATAT

ATC CAT

CAT

GTGGCA GTGGCA

TRT ATC

TCA GTGCCATAT

GGCATAT GTGGCATA

CAT

GGCATATC AGAGGATA TGGC AC GGCATAT

CCCAT

AT CCCAC CCAT ATCCCGG

CCGAT AT

GCCCTCTC CAT

TT c GA

06 2 ATCGGAGGGATACGAG

ATCGGAGG‘ ATCGGAGGGRTACG

ATCGGAGGGATACGAG ATCGGAGGGATACGAG ATCGGAGGGATRCGAG ATCGGAGGGATAC ATCGGAGGGATACG

TCGGAGGGATACGAC GAGGGATACGRG

N3 JS 1 CT *cc GAC AA*

CTAAG AGG CGG TG

TCCAG CTAGA

CT *cc GAC ARA xl

CT ACC GAC &AR CT ACC GAC AAA

*cc GAC AA* RCC GAC AM

CT *cc GAC AA* *cc GRC AAA *cc GAC AA* AK CAC AA*

cc GAC AA* CT *cc GAC AA* CT RCC GAC AAA CT *cc GAC AA*

C GAC AA.4

CT RCC GAC AAA X8

TA CT RCC GAC WA

c GAC AAA

CCG CC GAC AAA c cc GAC AA* TAGGG cc GAC AAA TTG CT RCC GAC AAA GGAG CT *cc GAC AA* CTGCT T RCC GAC AAA TCCC CT *cc GAC AA*

CC GAC AM cc *cc GAC AA* CTCGGTTTCCT *cc GAC AA* RGAGAAAA AK GAC AAA CTCTG CT *cc GAC AAA G CT AK GAC AM GGG cc GAC AA*

CT ACC GAC RRA

Figure 6. The Pattern of BID Expression in Various Lymphocyte Populations indicates That the Expansion of BID+ T Cells Occurs Mainly Extrathy mically

Junctional region sequences of translatable cDNA clones are presented for the various groups indicated. RPLs were isolated from a-week-old athymic BALBlc and C57BU6 mice; thymocytes were isolated from 4-week-old BALB/c mice; and systemic lymph nodes were isolated from 30-week-old BALBlc mice.

The existence of BID-like sequences in BALBIc suggests that the BID family is selected in this environment. One could not formally rule out the remote possibility that the BID rearrangement cannot be made in the C57BU6 strain due to some unknown molecular impediment. However, all the germline elements are present and are being used in C57BU6; they are complete, they can be rearranged, and C57BU6 mice can perform “fetal-type” rearrange- ments as demonstrated by their dECs. The predominance of BID in Fl(C57BU6 x BALB/c), BALB.B, BALB/c nulnu,

and in the lymph nodes of old BALB/c mice indicates, for reasons previously discussed, that the dominant selec- tion of this phenotype occurs essentially in the periphery. This is distinct from the positive selection of the up clono- types, which occurs in the thymus (Kisielow et al., 1968; Sha et al., 1988; Kaye et al., 1989; Berg et al., 1989). One might speculate that the participation of selective pres- sures that cannot be connected to classical class I or class II MHC antigens is the cause for this new type of strain- specific T cell expansion. Whether this observation might be applicable to other specific yZi or up receptors is yet to be determined. Nevertheless, the long-lasting expansion of BID+ T cells in BALB/c mice suggests that while pro- liferating throughout the life of the animal, such cells might perform a specific function.

Experimental Procedures

y6 T Cell Preparations Lungs from normal and nude mice were extensively perfused to re- move circulating blood, dissected, and the CD4-CD6- double nega- tive (DN) RPLs were purified from the dissected tissue according to our published protocol (Augustin et al., 1969). Cells were cultured for 72 hr in a mixture of lymphokines essentially as described, except that PMA and ionomycin were included only in the first 24 hr. The viable cells were separated from the dead cells by ficoll-hypaque centri- fugation, and ap+ T cells were removed by treatment with biotin-con- jugated H5%597 (anti-@ TCRs, gift of R. Kubo) monoclonal antibody folloWed by streptavidin-coupled magnetic Dynal beads in conjunction with a Dynal magnetic particle concentrator. Typically, l-2 x lo6 cells were obtained starting from a pool of five mice. y6 T cells from 4-week- old BALB/c thymi were enriched from a fresh thymbcyte cell suspen- sion after the preparation of DN cells and the removal of up T cells as described for RPLs. Similar treatments were used to isolate y6 T cells from dissected lymph nodes after B cells were first removed by adher- ence to a rabbit anti-mouse immunoglobulin-coated dish (panning).

Nucleic Acids Total cellular RNA was extracted from the various y6 T cell prepara- tions by the APGC procedure (Chomczynski and Sacchi, 1967). Trace amounts of contaminating DNA were further removed by treatment with RNAase-free DNAase (Boehringer Mannheim).

cDNA synthesis was performed in a 20 ~1 reaction volume, starting from RNA extracted from lo6 cells. Six units of placenta ribonuclease inhibitor (BRL) and 200 units of cloned Moloney virus reverse transcrip- tase (BRL) were included in the reaction, together with 10 pmol of a

Cell 404

CS primer (5’CGAATTCCACAATCTTCTTG-3’). After 1 hr at 37oC, the reaction mixture was heated at 95’X for 2 min, and 1 nl was added directly to a PCR for amplification.

PCR PCRs were performed using a Perkin-Elmer-Cetus DNA Thermer Cy cler and Taq polymerase (Saiki et al., 1988). Both 5’and 3’primers were present at 0.5 pM and were tested with cloned DNA. The 3’ Cs primer for the PCR is 5’AACAGATGGTTTGGCCGGAG-3’ and is internal to the Cs primer used for cDNA synthesis. The 5’ primers are

Vs,: GGGATCCTGCCTCCTTCTAC Vss: GCTCATGGTGACTTCATCTC Vs3: TTCCTGGCTATTGCCTCTGAC Vs4: CCGCTTCTCTGTGAACTTCC Vss: TCCACTGACCAGACAGTGGC Vss: TCAAGTCCATCAGCCTTGTC V&T: CGCAGAGCTGCAGTGTAACT.

All primers are listed 5’-3’; primer sequences are derived from Chien et al. (1987a, 1987b), Elliott et al. (1988) and Takagaki et al. (1989a). The V&s primer hybridizes to all members of the Vss family (Chien et al., 1987b). Each PCR cycle consists of incubations at 94OC for 30 s, 55OC for 30 s, and 72OC for 3 min. Before the first cycle, the reaction mixture was denatured at 94OC for 1 min. After the last cycle, the incu- bation at 72oC was extended for another 6 min. Each Vs primer was used in an independent PCR in conjunction with the inner C&, primer. For each Vr, reaction, a cDNA minus control was set up in parallel. For cloning purposes, 20 cycles of PCR were performed, while reaction products obtained after 12 cycles of amplification were used in the Southern analysis.

Southern Blot Analysis of V6 Gene Expression One-twentieth of the cDNA reaction was used directly in each of the seven V,+pecific PCRs. Ten microliters of the PCR products was withdrawn after every third cycle, incubated for another 3 min at 72oC, and half of it was fractionated on a 1.5% agarose minigel to determine the minimum number of reaction cycles needed for the most abundant PCR product to become visible by ethidium bromide staining. At this point, the DNA was transferred onto a GeneScreen Plus membrane (New England Nuclear), prehybridized at 45OC in 6x SSC, 1% SDS, 10x Denhardt’s solution, 250 pglml denatured salmon sperm DNA, and 50 pglml yeast tRNA for 4 hr. Hybridization was performed under the same conditions, except that 1 pmollml of szP end-labeled Jr,,- or Jsz-specific oligonucleotides was included as probe for each of the two duplicate filters (J&t: 5’TTCCACAGTCACTTGGGTTCX JQ; 5’-CTC- CACAAAGAGCTCTATGC-3’). After 16 hr, the filters were washed in 1% SDS, 6x SSC, 20 mM sodium phosphate (pH 7.4) and 0.05% sodium pyrophosphate twice at room temperature for 10 min each and then for 15 min at 45OC before autoradiography.

DNA Sequencing PCR products were gel purified and the appropriate size fragments cloned into the Smal site of pUC18. DNA sequencing was performed on double-stranded plasmid DNA by the dideoxy method using Se- quenase (United States Biochemical Corp.).

Acknowledgments

We are indebted to C. Steinberg for help with the statistical analysis. We thank C. Steinberg and H. von Boehmer for critical comments and L. Angman for technical assistance. This work was supported in part by National Institutes of Health grants Al 19775 and Al 22295 to A. A. The Base1 Institute for Immunology was founded and is supported by F Hoffmann-La Roche & Co. Ltd., Basel, Switzerland.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adverrisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 2, 1990

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